The purpose of these studies is to establish a better understanding of the energy metabolism of biological tissues, in vivo. Towards this goal, the laboratory concentrates on the use of minimally-invasive techniques to evaluate cellular energy metabolism of heart and skeletal muscle as well as screening approaches in proteomics and post-translational modifications. The following major findings were made over the last year: 1) In vivo NADH fluorescence is a valuable tool in monitoring the energetics of individual cells. The cellular NADH fluorescence is believe to be primarily from the mitochondria, but the precise NADH pool contributing to the fluorescence is poorly defined. Using native gel approaches for separating functional protein complexes, we have found that Complex 1 enhances NADH fluorescence within the native gels when studied under anaerobic conditions. Further fractionation and isolation suggests that the NADH binding site is directly in Complex 1 may represent the important entry step of NADH into the initial steps of oxidative phosphorylation. This not only explains the excellent correlation of NADH fluorescence signals with metabolic activity, but provides direct insight into the interaction of NADH at this critical step in oxidative phosphorylation. 2) Protein phosphorylation in the mitochondrial matrix is beginning to be appreciated as a more wide spread mechanism of regulating mitochondrial energy metabolism. A 32P screen of the mitochondrial matrix phospho-protein turnover has been expanded to evaluate the role of different experimental conditions on the rate of 32P incorporation into the matrix proteins. Novel new observations include the presence of protein auto-phosphorylation reminiscent of bacterial two component systems. These auto-phosphorylation systems include creatine kinase, cytochrome oxidase, and the beta subunit of the F1-ATPase. We have also gained evidence for an extensive allosteric binding of phosphate metabolites in the matrix that may also play a significant role in enzymatic regulation in addition to the conventional phosphorylation reactions. 3) Minimally invasive, two photon excitation fluorescence microscopy (TPEFM) is being used to study sub-cellular metabolic processes within cells, in intact animals, under normal in vivo conditions using various exogenous and intrinsic fluorescent probes. We have continued to make improvements in the technology of this approach by modifying the telescoping system to permit rapid (KHz) focusing at the objective permitting the accurate collection of image plane stacks, as well as track image planes within the animal. Motion compensation algorithms have been also developed to compensate for in-plane displacements resulting in higher signal to noise performance. Using these approaches studies on the sub-cellular consequences of hypoxia, vaso-dilatation, work and insulin infusions are currently being investigated. 4) Using TPEFM we have defined the macromolecular structure of the arterial wall of the porcine coronary and murine aorta. These data reveal, for the first time, the full 3 dimensional microstructure of collagen and elastin in these structures. Using these data we proposed that the macromolecular structure is responsible for the early deposition of LDL in atherosclerosis in the susceptible regions around branch points and the aortic arch. Using fluorescently labeled LDL, we found that LDL indeed preferentially bound to these regions of the vasculature in a fashion consistent with an interaction with proteoglycans associated with exposed collagen at these sensitive sites. These data suggest that the macromolecular structure itself might be partially responsible for the initiation of the atherogenesis.